RETINOIC ACID RECEPTOR AND SP-B GENE REGULATION

本工作的长期目标是研究维甲酸(RA)及其受体(RAR)通过调控呼吸道上皮细胞中肺特异基因的表达来调节胎肺发育的功能作用，并将这一知识应用于临床治疗。表面活性蛋白B(SP-B)通过增加磷脂在空气-水界面的扩散速度来维持肺泡的稳定性，因此对出生后呼吸适应和氧化损伤后呼吸上皮的恢复至关重要。在这一应用中，我们建议确定RA和RAR在体外和体内刺激SP-B基因的分子机制。具体目的是：1)鉴定SP-B启动子上的顺式维甲酸反应元件(RARs)。将进行DNA足迹、凝胶电泳迁移率改变分析和定点突变。这些研究将在呼吸道上皮细胞中进行，包括H441细胞系、ML3-15细胞系和肺泡II型上皮细胞的原代分离株；2)RAR与组织特异性转录因子TTF-1相互作用的特征。将通过免疫共沉淀、Western印迹和酵母双杂交系统确定RAR和TTF-1的精确相互作用结构域，以及它们在结合和激活SP-B基因中顺式作用元件中的作用；3)顺-REARE的鉴定，以确定RA/RAR对转基因小鼠SP-B基因时空表达的影响。野生型和罕见突变的HSP-B启动子将与猫报告基因相连，并被引入老鼠体内。来自转基因肺的组织切片原位杂交和CAT检测将被用于评估体内罕见的组织切片杂交，来自转基因肺的CAT将被用于评估在体内决定细支气管和肺泡II型上皮细胞SP-B启动子表达水平和特异性的罕见功能。

The long term goals of this work are to study functional roles of retinoic acids (RA) and their receptors (RAR) in regulating fetal lung development by controlling lung specific gene expression in respiratory epithelial cells, and to apply the knowledge to clinical treatment. Surfactant protein B (SP-B) maintains alveolar stability by enhancing the rate of spreading of phospholipid at the air-water interface, and therefore is critical to postnatal respiratory adaptation and recovery of respiratory epithelium following oxidant injury. In this application, we propose to determine molecular mechanisms by which RA and RAR stimulate the SP-B gene both in vitro and in vivo. The specific aims are: 1) Identification of cis-retinoic acid responsive elements (RAREs) on the SP-B promoter. DNA footprint, gel electrophoresis mobility shift assay and site-specific mutagenesis will be performed. The studies will be done in respiratory epithelial cells, including the H441 cell line, the ML3-15 cell line and primary isolates of alveolar Type II epithelial cells; 2) Characterization of the interaction between RAR and the tissue-specific transcription factor, TTF-1. Co-immunoprecipitation, Western blot and the yeast two-hybrid system will be performed to identify the precise interaction domains of both RAR and TTF-1, and their roles in binding and activating cis-acting elements in the SP-B gene; 3) Characterization of cis-RARE to determine the effects of RA/RAR on the temporal and spatial expression of the SP-B gene in transgenic mice. The wild type and RARE site-mutated hSP-B promoters will be linked to the CAT reporter gene and introduced into mice. In situ hybridization of tissue sections and CAT assay from transgenic lungs will be used to assess the in vivo RARE hybridization of tissue sections and CAT assay from transgenic lungs will be used to assess the in vivo RARE functional roles determining SP-B promoter expression levels and specificities in bronchiolar and alveolar Type II epithelial cells.